WO2018016814A1

WO2018016814A1 - Highly stable fibroblast growth factor variant having modified n-terminal amino acid region, and use thereof

Info

Publication number: WO2018016814A1
Application number: PCT/KR2017/007637
Authority: WO
Inventors: 신항철; 오종광; 박연희
Original assignee: (주)피앤피바이오팜
Priority date: 2016-07-21
Filing date: 2017-07-17
Publication date: 2018-01-25
Also published as: KR20180010468A; KR101891678B1

Abstract

The present invention relates to a method for inhibiting the degradation of a highly stable bFGF in a production process, using a microorganism, by modifying the N-terminal amino acid sequence of the bFGF, thereby increasing productivity. More specifically, provided are: a variant in which N-terminal deletion and insertion mutations are induced in the highly stable bFGF variant in which, in an amino acid sequence of SEQ ID NO: 1, two or more amino acids are substituted with serine and one or more amino acids are substituted with cysteine; a DNA base sequence coding for the bFGF variant; an expression vector comprising the DNA base sequence; a transformant transformed by the expression vector; a method for producing the bFGF variant; and a composition containing the bFGF variant as an active ingredient. According to the present invention, the bFGF variant of the present invention has excellent thermal stability and aqueous solution state stability, and thus a functional cosmetic product and a skin inflammation treatment agent, which unlike conventional native bFGF products do not lose activity even during distribution and storage processes, can be produced.

Description

Highly stable fibroblast growth factor variants having modified N-terminal amino acid sites and uses thereof

The present invention relates to a highly stable fibroblast growth factor variant having a modified N-terminal amino acid site and its use, and more particularly, to an N-terminal amino acid of a basic fibroblast growth factor (bFGF). Through modification of the sequence, the present invention relates to the production of pure single component high stability bFGF by inhibiting the N-terminal amino acid loss that may occur during production using microorganisms.

Growth factors play an important role in regulating cell growth, proliferation and differentiation. Therefore, there is a system that naturally repairs the damage and aging of the skin caused by internal and external factors such as wounds and surgery, and growth factors play an important role here. In order to maintain the function of each organization, various growth factors are generated and maintained at a constant concentration. As age increases, the concentration of growth factors in each tissue, such as the skin, is lowered, and aging progresses, as the regeneration and division of cells are weakened, wrinkles are formed, and elasticity is decreased.

Among them, bFGF is composed of 154 amino acids and is composed of a polypeptide having a molecular weight of 17,123 Daltons. It plays an important role in development, angiogenesis and wound healing. bFGF is a mitogen and chemotactic factor and is known to be a powerful mediator of wound healing, angiogenesis, and growth of the nervous system.

However, the growth factors present in these blood and tissues are known to have very short half-lives in the body, such as several minutes. In particular, bFGF has four cysteine residues that do not form disulfide bonds in its structure. There is a problem that is greatly affected by the stability of having.

In addition, the bioavailability of protein therapeutics such as bFGF is often limited by short blood half-lives and susceptibility to proteases, which interfere with maximum clinical efficacy. In order to improve the use of bFGF more effectively, the physical and chemical stability in vitro should be improved in addition to the stability in the body, and thus the use of bFGF will be increased in the manufacture, storage, and distribution of pharmaceuticals and cosmetics. In order to improve this problem, P & P Biofarm developed high stability bFGF (named HsbFGF) in 2016 (KR 10-2015-0078930 / PCT-KR2015-007734).

However, when high stability bFGF is produced using Escherichia coli, the N-terminus of bFGF is partially hydrolyzed by aminopeptidase, resulting in the loss of some amino acids at the N-terminus of bFGF. As a result, the high-purity bFGF final purified product has a mixture of different N-terminus, and is difficult to separate.

In other words, the N-terminal heterogeneity problem is misunderstood as an impurity, that is, it is recognized as not a single component, which interferes with the production and quality control of the recombinant protein and must be solved because it is recognized as a low-purity product in the drug licensing process. This is a task that must be done (KFDA B1-2014-3-018).

SUMMARY OF THE INVENTION The present invention has been made in view of the above problems and the above-mentioned necessity, and an object of the present invention is to provide a highly stable bFGF N-terminal variant capable of successfully inhibiting N-terminal amino acid loss.

Another object of the present invention is to provide a DNA base sequence encoding a bFGF variant.

In addition, another object of the present invention is to provide an expression vector comprising the DNA base sequence.

In addition, another object of the present invention is to provide a transformant transformed by the expression vector.

Another object of the present invention is to provide a method for producing high stability bFGF variant.

In addition, another object of the present invention to provide a cosmetic composition for improving skin conditions comprising a high stability bFGF variant as an active ingredient.

In addition, another object of the present invention to provide a pharmaceutical composition for preventing or treating skin diseases comprising a high stability bFGF variant as an active ingredient.

In order to achieve the above object, the present invention provides a highly stable bFGF wherein two or more amino acids in the amino acid sequence of SEQ ID NO: 1 is substituted with serine, one or more amino acids are substituted with cysteine, and one amino acid on the surface is substituted with tyrosine. Provide variants.

More preferably, the recombinant protein to be finally produced by introducing a deletion mutation at two amino acid residues of the N-terminal region of the existing high stability bFGF variant and inserting methionine to prevent N-terminal hydrolysis that may occur immediately after expression of the recombinant protein. It was to have a homogeneity of.

As used herein, the term bFGF is a basic protein (pI 9.58) with a molecular weight of about 18 kDa, which is mainly secreted by the pituitary gland and is known to promote growth of various mesodermal derived cells. In addition, it is a protein that promotes the growth of vascular endothelial cells and smooth muscle cells, has an excellent effect on the treatment of trauma and vasculature, increases the synthesis of collagen and elastin, maintains skin elasticity, helps normal cells grow, and It is known to promote the recovery and to perform the healing action.

Variants of the present invention, through the homogeneous alignment (homology alignment) method of the tertiary structure and species of bFGF and computer molecular protein modeling, by selecting a site unrelated to the active site of bFGF, was prepared through mutation experiments As a variant, cysteine amino acid residues in which bFGF and other bFGFs form disulfide bonds are replaced with serine residues of similar structure to increase stability against precipitation due to disulfide bonds on the surface. In addition, stability is increased by a method of reducing loop entropy by additionally generating disulfide bonds by replacing one residue close to the loop in bFGF with cysteine. In addition, by replacing histidine residues with tyrosine in bFGF, hydrogen bonds and van der Waals interactions are increased to stabilize the cavity structure inside the protein structure.

According to a preferred embodiment of the invention, the serine-substituted amino acids are the 68th cysteine and 86th cysteine in the amino acid sequence of SEQ ID NO: 1.

According to a preferred embodiment of the present invention, the amino acid substituted with cysteine is 25 lysine in the amino acid sequence of SEQ ID NO: 1; The 33rd isoleucine at the amino acid sequence of SEQ ID 1; The 39th valine in the amino acid sequence of SEQ ID 1; The 49th histidine in the amino acid sequence of SEQ ID 1; The 51st lysine in the amino acid sequence of SEQ ID 1; Alanine at the amino acid sequence of SEQ ID 1; The 75th methionine in the amino acid sequence of SEQ ID 1; The 116th alanine in the amino acid sequence of SEQ ID 1; The 66th glycine in the amino acid sequence of SEQ ID 1; 67th valine in amino acid sequence of SEQ ID 1; The 69th alanine in the amino acid sequence of SEQ ID 1; 81 leucine in the amino acid sequence of SEQ ID 1; Alanine at the amino acid sequence of SEQ ID 1; 107 th serine in the amino acid sequence of SEQ ID 1; The 135 th alanine in the amino acid sequence of SEQ ID 1; The 136th isoleucine in the amino acid sequence of SEQ ID 1; 137 leucine in the amino acid sequence of SEQ ID 1; And at least 143 alanine in the amino acid sequence of SEQ ID NO: 1, and more preferably 39 th valine in the amino acid sequence of SEQ ID NO: 1; The 49th histidine in the amino acid sequence of SEQ ID 1; The 51st lysine in the amino acid sequence of SEQ ID 1; Alanine at the amino acid sequence of SEQ ID 1; The 75th methionine in the amino acid sequence of SEQ ID 1; At least one member selected from the group consisting of the 116th alanine in the amino acid sequence of SEQ ID NO: 1, and most preferably the 74th alanine in the amino acid sequence of SEQ ID NO: 1.

As an additional variant, the variant in which the histidine No. 49 residue, which is a residue exposed on the surface of the protein, is substituted with tyrosine in the variant where the 74th alanine is substituted with cysteine, the most preferred variant. That is, the bFGF variant of the present invention is the 68th and 86th amino acid residues of the variant (SEQ ID NO: 1) after removing two amino acid sequences of the N-terminal sequence from the wild-type and adding methionine as a starting sequence. All cysteines are substituted with serine, the 49th histidine is substituted with tyrosine, and the 74th amino acid residue, alanine, is further substituted with cysteine to form intramolecular disulfide bonds and the remaining amino acid sequence is identical to the naturally occurring amino acid sequence. Provide variants.

The bFGF variant of the present invention is a variant made by inducing a deletion mutation of the N-terminal sequence to the sequence of the aforementioned HsbFGF (KR 10-2015-0078930 / PCT-KR2015-007734).

More preferably, the first and second sequences of proline and alanine at the N-terminus of the existing HsbFGF are induced as deletion mutations and methionine is inserted as a start sequence.

The bFGF variant of the present invention has increased heat stability compared to the native form while maintaining protein activity. As shown in Experimental Example 2 below, the bFGF variant has the same activity as the natural type, and it can be seen that the stability against heat was also significantly increased. Among the bFGF variants, amino acids 68 and 86 were substituted with serine, amino acid 74 was substituted with cysteine, and the variant K (sbFGF, K74) of the present invention, which induced disulfide bonds, was substituted with tyrosine of histidine No. 49 in variant K. In the case of variant O (HsbFGF), the thermal stability was improved than that of the natural type control group.

According to another aspect of the present invention, the present invention provides a DNA base sequence (SEQ ID NO: 2) encoding the bFGF variant and an expression vector comprising the same.

Expression vectors of the present invention can be prepared by inserting the gene of bFGF in a general expression vector. In a preferred embodiment of the present invention, pET21a vector was used as the expression vector, but the present invention is not limited thereto, and any cell expression vector that can be used generally can be used. In a preferred embodiment of the present invention, a vector in which the bFGF gene was inserted into the pET21a vector was prepared and named "pSSB-bFGF" (FIG. 2).

According to another aspect of the present invention, the present invention provides a transformant which is a host cell transformed by the expression vector.

The bFGF variant of the present invention can be prepared by transforming a host cell with a vector containing a gene encoding a bFGF variant prepared by site-specific mutation induction and the like, and expressing the bFGF variant by a chemical amino acid synthesis method. Can be prepared.

Preferred as the DNA sequence encoding the bFGF variant is the 68th and 86th codons with the removal of the N-terminal proline and alanine from the native bFGF and the insertion of methionine in place with the codons encoding serine and the 74th codon It was substituted with a codon encoding this cysteine. In addition, in the case of variant O (HsbFGF) substituted with tyrosine of histidine No. 49 of the variant K, the thermal stability superior to the natural type and the variant K (sbFGF) was improved.

On the other hand, since many codons encoding one amino acid exist due to degeneracy of codons, it is well known that even if DNAs encoding the same amino acid sequence have different nucleotide sequences. Such DNA encoding the bFGF variant may be chemically synthesized or prepared by using a method such as site-specific mutation induction based on the production of native bFGF cDNA.

The DNA encoding the bFGF variant of the present invention prepared above can be expressed using any of the appropriate prokaryotic or eukaryotic expression systems well known in the art (Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd). ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, USA, 1989). Expression is preferably performed in Escherichia coli, eg, Escherichia coli BL21 (DE3), Escherichia coli JM109 (DE3), Escherichia coli NM522, etc. for unglycosylated bFGF variants, and suitable vectors that can be used for expression in Escherichia coli are Sambrook (Homologous) and Fiers et al. ("Proced. 8th Int. Biotechnology Symposium", Soc. Frac. De Microbiol., Paris, (Durand et al., Eds.), Pp. 680-697 , 1988).

Transformation of host cells by the vector described above can be carried out by any of the conventional methods (Sambrook et al., Molecular Cloning, A Laboratory Manual, 1989; Ito et al., J. Bacteriol. 153: 263 , 1983). When transforming Escherichia coli, water-soluble cells capable of absorbing DNA can be prepared, and then treated according to known methods.

According to another aspect of the present invention, the present invention provides a method for producing high stability bFGF variant, comprising the steps of: (a) culturing the transformant; And (b) separating the variant from the culture obtained in step (a).

According to a preferred embodiment of the present invention, step (b) of the method comprises the steps of: (c) cell disrupting the transformant and removing aggregates; (d) separating and purifying the supernatant from which the aggregates have been removed using ion exchange resin chromatography; And (e) separating and purifying the highly stable bFGF variant after the ion exchange resin using heparin affinity chromatography.

In general, host microorganisms containing the desired expression vector are cultured at their optimum growth conditions in a range that maximizes the production of the desired protein. For example, E. coli BL21 (DE3) cells transformed with a vector containing an empicillin resistance gene as a selection marker are cultured at 37 ° C in LB medium containing empicillin.

Recovery and purification of the produced bFGF variant after culturing the transformed host cell can be carried out by using various methods known in the art or a combination thereof. For example, bFGF variants expressed in transformed E. coli cells can be recovered by extraction from cell culture or by appropriate methods known in the proteomics following cell disruption.

Preferably, to purify the bFGF variant, the culture medium of recombinant E. coli cells is centrifuged to harvest the cells, and the harvested cells are suspended in lysozyme-added buffer and crushed by ultrasound. The cell lysate is centrifuged to remove aggregates in insoluble granules. The supernatant from which the aggregates are removed is separated and purified using ion exchange resin chromatography, and purified by ion exchange resin followed by heparin affinity chromatography to obtain the resultant high stability bFGF variant.

According to another aspect of the present invention, the present invention provides a pharmaceutical composition for preventing or treating skin diseases comprising the high stability bFGF variant as an active ingredient.

As demonstrated in the examples below, the high stability bFGF variants of the present invention have the same activity as native bFGF, and have very good thermal stability and stability in aqueous solution. Therefore, the composition of the present invention is very effective for the prevention or treatment of skin diseases. Preferably, the composition of the present invention is such as skin inflammation, acute and chronic eczema, contact dermatitis, atopic dermatitis, seborrheic dermatitis, chronic simple gland, interrogation, deprivation dermatitis, papular urticaria, psoriasis, sun dermatitis and acne. Used for the prevention or treatment of skin diseases.

In addition, the composition of the present invention can provide a composition for treating wounds. Preferably, the composition of the present invention is used for the treatment of closed wounds and open wounds. Examples of closed windows include contusion or bruise, and examples of open windows include abrasion, laceration, avulsion, penetrated wound and gun-shot wound. .

The composition of the present invention comprises (a) a pharmaceutically effective amount of the bFGF variant of the invention described above; And (b) a pharmaceutically acceptable carrier.

As used herein, the term “pharmaceutically effective amount” means an amount sufficient to achieve the efficacy or activity of the bFGF variants described above.

Pharmaceutically acceptable carriers included in the pharmaceutical composition of the present invention are commonly used in the preparation, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, silicic acid Calcium, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oils, and the like. It is not. In addition to the above components, the pharmaceutical composition of the present invention may further include a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition of the present invention may be administered orally or parenterally, preferably parenterally, and in the case of parenteral administration, it may be administered by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, topical administration, transdermal administration, or the like. Can be.

Suitable dosages of the pharmaceutical compositions of the present invention may vary depending on factors such as the formulation method, mode of administration, age, weight, sex, morbidity, condition of food, time of administration, route of administration, rate of excretion and response to response of the patient. Can be. Meanwhile, the preferred daily dose of the pharmaceutical composition of the present invention is 0.001-100 mg / kg.

The pharmaceutical compositions of the present invention may be prepared in unit dose form by formulating with a pharmaceutically acceptable carrier and / or excipient according to methods which can be easily carried out by those skilled in the art. Or may be prepared by incorporation into a multi-dose container. In this case, the formulation may be in the form of a solution, suspension, or emulsion in an oil or aqueous medium, or may be in the form of extracts, powders, granules, tablets, capsules, or gels (eg, hydrogels), and may further include dispersants or stabilizers. .

According to another aspect of the present invention, the present invention provides a cosmetic composition for improving skin condition comprising the high stability bFGF variant as an active ingredient.

As demonstrated in the examples below, the high stability bFGF variants of the present invention have the same activity as native bFGF, and have very good thermal stability and stability in aqueous solution. Therefore, the composition of the present invention is very effective for improving skin condition.

Preferably, the composition of the present invention is used to improve skin conditions such as wrinkle improvement, skin elasticity improvement, skin aging prevention, hair loss prevention or hair growth, skin moisturization improvement, mushroom removal or acne treatment.

The composition of the present invention comprises (a) a cosmetically effective amount of the bFGF variant of the present invention described above; And (b) a cosmetically acceptable carrier.

As used herein, the term "cosmetic effective amount" means an amount sufficient to achieve the skin improving efficacy of the composition of the present invention described above.

Cosmetic compositions of the present invention may be prepared in any formulation conventionally prepared in the art, including, for example, solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, soaps, surfactant-containing cleansing , Oils, powder foundations, emulsion foundations, wax foundations and sprays, and the like, but are not limited thereto. More specifically, it may be prepared in the form of a flexible lotion, nutrition lotion, nutrition cream, massage cream, essence, eye cream, cleansing cream, cleansing foam, cleansing water, pack, spray or powder.

When the formulation of the present invention is a paste, cream or gel, animal oils, vegetable oils, waxes, paraffins, starches, trachants, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicas, talc or zinc oxide may be used as carrier components. Can be.

When the formulation of the present invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used, in particular in the case of a spray, additionally chlorofluorohydrocarbon, propane Propellant such as butane or dimethyl ether.

When the formulation of the present invention is a solution or emulsion, a solvent, solubilizer or emulsifier is used as the carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 Fatty acid esters of, 3-butylglycol oil, glycerol aliphatic ester, polyethylene glycol or sorbitan.

When the formulation of the present invention is a suspension, liquid carrier diluents such as water, ethanol or propylene glycol, suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, microcrystals Soluble cellulose, aluminum metahydroxy, bentonite, agar or tracant and the like can be used.

When the formulation of the present invention is a surfactant-containing cleansing, the carrier component is aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyltaurate, sarcosinate, fatty acid amide. Ether sulfates, alkylamidobetaines, aliphatic alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives or ethoxylated glycerol fatty acid esters and the like can be used.

The components included in the cosmetic composition of the present invention include components conventionally used in cosmetic compositions, in addition to the bFGF variant and carrier component as active ingredients, and include, for example, conventional agents such as antioxidants, stabilizers, solubilizers, vitamins, pigments and flavorings. Phosphorus adjuvant.

Since the compositions of the present invention include the above-mentioned high-stability bFGF variant of the present invention as an active ingredient, the contents common between the two are omitted in order to avoid excessive complexity of the present specification.

According to the present invention, the novel HsbFGF variant made through the modification of the N-terminus has excellent thermal stability and stability in aqueous solution, and thus does not lose activity unlike conventional natural bFGF products during distribution and storage, and when produced in microorganisms, It is made of pure single substance and can be used as a material for functional cosmetics and as a therapeutic agent for skin wounds.

1 and 2 show an overview of the assembly of plasmid pSSB-bFGF.

Figure 3 is an analysis using SDS-PAGE after the final purification of the native type bFGF and the existing K75 (sbFGF) and the novel HsbFGF (Variant O) developed in the present invention.

Figure 4 shows the results of the difference between Tm (melting temperature) which is an indicator of the thermal stability of the natural type of bFGF and K74 (variant K), the existing HsbFGF (existing variant) and the new HsbFGF (variant O).

Figure 5 shows the results of stability after incubation for 5 days at 50 ℃ in phosphate buffer of the natural type bFGF and the existing HsbFGF and novel HsbFGF (Variant O).

Figure 6 shows the results of the stability after incubation for 5 days at 60 ℃ in phosphate buffer of the natural type bFGF and the existing HsbFGF and novel HsbFGF (Variant O).

Figure 7 shows the comparison between the natural type of bFGF and the novel HsbFGF (Variant O) of the present invention and the existing HsbFGF activity.

8 shows the N-terminal analysis of the novel HsbFGF (Variant O) of the present invention, Figure 9 shows the N-terminal analysis of the existing HsbFGF.

10 is a result of analyzing the results of incubation for one month at 37 ° C. of natural bFGF, existing HsbFGF variant, and novel HsbFGF (variant O) by concentration quantification.

11 and 12 show the results of incubation at 37 ° C. for 100 days with natural bFGF and K74 (variant K), and novel HsbFGF (variant O) of the present invention using SDS-PAGE and HPLC.

Figure 13 shows the results of toxicity experiments using L929 cells of novel HsbFGF (Variant O).

Hereinafter, the embodiments are only for illustrating the present invention in more detail, and the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention, those skilled in the art to which the present invention pertains. Will be self-evident.

Experiment Method and Materials

DNA construction

PET21a (Fig. 1), which is a protein expression vector, and BL21 (DE3) were purchased from Novagen as the E. coli strain for expression, and Top10 was used as the cloning E. coli strain. Restriction enzymes used for genetic recombination are all NEB (New England Biolabs) product, the ligase is Roche's T4 DNA ligase. Ex taq DNA polymerase used in PCR is a product of Takara and pfuUltra used for point mutation HF DNA polymerase is a product of Agilent. DNA gel extraction kit and plasmid mini prep kit are products of Cosmojintech. In addition, primers were produced by Cosmojin Tech Co., Ltd., DNA sequencing was also performed by Cosmojin Tech Co., Ltd.

Protein expression

The expression derivative IPTG (isopropyl-1-thio-β-D-galactopyranoside) and ampicillin and chloramphenicol used as antibiotics were all purchased from Sigma. E. coli The bacto tryptone and yeast extract used to make cultured LB medium were purchased from BD (Becton Dicknson), and NaCl was used as a Duksan product.

Protein purification

Reagents used in the purification process using the highest purity products, and the reagents used in the purification process are as follows. sodium phosphate monobasic (Sigma), sodium phosphate dibasic (Sigma), sodium chloride (Sigma). The columns used in FPLC were SP-sepharose and heparin affinity columns.

FPLC

FPLC used GE UPC-800.

CD (Circular dichroism )

The CD used Jasco's J-810 spectropolarimeter.

Homology modelling Homology modeling

Homology modeling used Modeller (Andrej Sali).

Energy minimization

Energy minimization was performed using the Amber 99FF force field included in Chimera.

Disulfide bonds Disulfide predict

The YASARA Web server was used to predict the formation of disulfide bonds.

Disulfide bonds Distance measurement

The plotting program for measuring disulfide bond distances was performed using protein contact map visualization (Andreas Viklund).

Point mutation

In order to increase the stability of bFGF, the amino acid moiety to be changed was found through the structure of the protein (PDB: 4FGF) and the molecular model method, and amplified by the Quickchange mutagenesis method using pfuUltra HF DNA polymerase with the following primer (Example 2). . In order to remove the used bFGF template, the DpnI reaction was performed and transformed to Top10, and mutants were identified through sequencing.

molecule modelling (Molecular modeling)

Candidate groups capable of disulfide bonds were set using 1BLA (NMR), the structure of proteins registered in PDB. The protein contact map visualization program was used to analyze residues with C-alpha carbon of less than 7Å and C-beta carbon of 5Å. Subsequently, the formation of disulfide bonds was analyzed using a Yasara energy minimization server, and energy minimization was performed using the AMBER force field FF99 of chimera. Then, the structure of the produced protein was aligned with the bFGF of the natural type, and the structure having a value of less than 0.5 was measured by RMSD.

CD (Circular dichroism )

For structural analysis and Tm measurement of native bFGF and variants, bFGF is constant to 0.2 mg / ml, respectively. And put into 0.1 cm cell, band width 1 nm, response 0.25 sec, data pitch 0.1 nm, scanning speed 20 nm / min, pathlength length 1 cm, accumulation No. 8, temperature at 20 ℃ Was analyzed. To analyze the temperature stability, the melting temperature was performed at a concentration of 0.2 mg / ml in a 0.1 cm cuvette at 205 nm wavelength at 20 ℃ and 95 ℃. The conditions were measured from 20 degreeC to 95 degreeC on condition of 1 degree-C / min.

Cell proliferation assay

Experiments were performed using the cell proliferation ability to confirm that the naturally produced bFGF and the variants are actually active. NIH-3T3 and L929 cells used in the experiment were maintained using DMEM complete medium containing 10% heat-inactivated fetal bovine serum, 100 units / ml penicillin, and 100 mg / ml streptomycin. 2x10 ³ cells / well of NIH-3T3 cells were seeded on a 96 well culture plate. The NIH-3T3 cells incubated for 24 hours were treated with sample solution in DMEM medium containing 0.5% FBS for each concentration after starvation with serum-free DMEM medium, and cultured for 72 hours. After incubation, 10 ul of MTT [3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl-2H-tetrazolium bromide] solution was added and reacted for 2 hours, followed by formazan crystal with 100 ul of DMSO. Was dissolved. Absorbance was measured at 540 nm using a spectrophotometer. Drug susceptibility was compared as a percentage of drug-treated wells for absorbance of untreated drug (control).

Incubation test

Incubation test of the native type bFGF and the variant was carried out to confirm the storage degree at room temperature. Each native bFGF and its variants were dissolved at 0.5 mg / ml in 1X PBS (pH 7.2) and incubated in 37 ℃, 50 ℃ and 60 ℃ water baths. After sampling for 24 hours, centrifugation at 13000 rpm for 15 minutes at 4 ℃ to obtain only the supernatant and quantitative analysis and HPLC analysis by nano drop.

Example 1 Construction and Purification of pSSB-bFGF Plasmids Containing Human bFGF cDNA

Human mononuclear cDNA library was used as a template, and DNA encoding bFGF was prepared by polymerase chain reaction using a primer. The base sequence of the primer used is as follows:

Sense primer: 5'-GGCGGGCATATGTTGCCCGAGG-3 '

Antisense Primer: 3'-CCTCGGGCAACATATGCCCGCC-5 '.

The bFGF portion of FIG. 2 was amplified using the primers specified above, and 1 ug of the amplified DNA fragment was dissolved in 50 ul TE (pH8.0) solution, followed by 2 units of NdeI (NEB) and 2 units of After mixing with BamHI (NEB), the reaction was carried out at 37 ℃ for 2 hours to have a NdeI restriction enzyme site at the 5'-end and BamHI restriction site at the 3'-end. After only DNA was purified using a DNA purification kit (GeneAll), 10 ul of 20 ng of this DNA fragment was treated with 20 ng of pET21a (+) plasmid prepared by NdeI and BamHI in the same manner. After mixing in a TE (pH 8.0) solution, 1 unit of T4 DNA ligase (NEB Co., Ltd.) was added thereto, and reacted at 16 ° C. for 4 hours for conjugation. The resulting plasmid was named pSSB-bFGF. The expression plasmid thus prepared was transformed into heat shock in E. coli BL21 (DE3). After transformation, colonies resistant to ampicillin in solid medium were selected and inoculated into 10 ml of LB medium (LB / ampicillin). The selected expression strains were incubated at 37 ° C. for 12 hours, mixed with 100% glycerol 1: 1, and stored at −70 ° C.

Expression strains were inoculated in 10 ml of LB medium (LB / ampicillin) and then cultured for 12 hours or more. Thereafter, 500 ml of LB medium (LB / ampicillin) was transferred to an absorbent OD _{600 of} 0.4 to 0.5, and IPTG (isopropyl-1-thio-β-D-galactopyranoside) was added to a final concentration of 0.5 mM. After shaking for 4 hours at 37 ℃ shaking culture at 200 rpm centrifuged at 8000 rpm for 10 minutes to obtain E. coli pellets (pellets) cells were disrupted by ultrasonic treatment. Then SP and heparin column purification using FPLC. After confirming the fraction containing bFGF for each fraction through the SDS-PAGE analysis, it was quantitated by Bradford assay method, and as a result 10 ~ 18 mg of bFGF was obtained.

Example 2: Construction of pSSB-bFGF Variant Plasmids

PfuUltra as a template using the native pSSB-bFGF plasmid PSSB-bFGF variant plasmids were made by PCR using two complementary primers corresponding to the respective variants using HF DNA polymerase. Variant plasmids were identified. The base sequence of the primer used is as follows:

Sense primer 5'-TCT ATC AAA GGA GTG TCT GCT AAC CGT TAC CTG-3 'and antisense primer 3'-CAG GTA ACG GTT AGC AGA CAC TCC TTT when the 68th cysteine codon TGT is substituted with serine codon TCT GAT AGA-5 ';

Sense primer 5'-TTA CTG GCT TCT AAA TCT GTT ACG GAT GAG TGT-3 'and antisense primer 3'-ACA CTC ATC CGT AAC AGA TTT AGA AGC when TGT, the codon of the 86th cysteine, is substituted by TCT, the codon of serine CAG TAA-5 ';

Senseprimer 5'-GCT AAC CGT TAC CTG TGC ATG AAG GAA GAT GGA-3 'and antisense primer 3'-TCC ATC TTC CTT CAT GCA CAG GTA ACG GTT AGC-5 ';

When the AAA, the codon of the 25th lysine, is substituted with TGC, the codon of cysteine, the sense primer 5'-AAG CGG CTG TAC TGC TGC AAC GGG GGC TTC TTC-3 'and the antisense primer 3'-GAA GAA GCC CCC GTT GCA GCA GTA CAG CCG CTT-5 ';

When the ATC, the codon of the 33rd isoleucine, is substituted with TGC, the codon of cysteine, the senseprimer 5'-GGC TTC TTC CTG CGC TGC CAC CCC GAC GGC CGA-3 'and the antisense primer 3'-TCG GCC GTC GGG GTG GCA GCG CAG GAA GAA GCC-5 ';

When the 39th valine codon GTT is substituted with cysteine codon TGC, the senseprimer 5'-CAC CCC GAC GGC CGA TGC GAC GGG GTC CGG GAG-3 'and the antisense primer 3'-CTC CCG GAC CCC GTC GCA TCG GCC GTC GGG GTG-5 ';

Senseprimer 5'-GAG AAG AGC GAC CCT TGC ATC AAG CTA CAA CTT-3 'and antisense primer 3'-AAG TTG TAG CTT GAT GCA AGG GTC GCT CTT CTC -5 ';

When the 51st lysine codon AAG is substituted with cysteine codon TGC, senseprimer 5'-AGC GAC CCT CAC ATC TGC CTA CAA CTT CAA GCA-3 'and antisense primer 3'-TGC TTG AAG TTG TAG GCA GAT GTG AGG GTC GCT-5 ';

When the ATG, the codon of the 75th methionine, is substituted with TGC, the codon of cysteine, the sense primer 5'-AAC CGT TAC CTG GCT TGC AAG GAA GAT GGA AGA-3 'and the antisense primer 3'-TCT TCC ATC TTC CTT GCA AGC CAG GTA ACG GTT-5 ';

Senseprimer 5'-ACC AGT TGG TAT GTG TGC CTG AAG CGA ACT GGG-3 'and antisense primer 3'-CCC AGT TCG CTT CAG GCA CAC ATA CCA when GCA, the codon of 116th alanine, is substituted with TGC, a codon of cysteine ACT GGT-5 ';

Sense primer 5'-GTT GTG TCT ATC AAA TGC GTG TCT GCT AAC CGT-3 'and antisense primer 3'-ACG GTT AGC AGA CAC GCA TTT GAT AGA when GGA, the codon of the 66th glycine, is substituted with TGC, the codon of cysteine CAC AAC-5 ';

GTG, the codon of the 67th valine, was substituted with TGC, the codon of cysteine Sense Primer 5'-GTG TCT ATC AAA GGA TGC TCT GCT AAC CGT TAC-3 'and Antisense Primer 3'-GTA ACG GTT AGC AGA GCA TCC TTT GAT AGA CAC-5 ';

Sense primer 5'-ATC AAA GGA GTG TCT TGC AAC CGT TAC CTG GCT-3 'and antisense primer 3'-AGC CAG GTA ACG GTT GCA AGA CAC TCC TTT GAT-5 ';

Senseprimer 5'-AAG GAA GAT GGA AGA TGC CTG GCT TCT AAA TCT-3 'and antisense primer 3'-AGA TTT AGA AGC CAG GCA TCT TCC ATC TTC CTT-5 ';

Senseprimer 5'-GAT GGA AGA TTA CTG TGC TCT AAA TCT GTT ACG-3 'and antisense primer 3'-CGT AAC AGA TTT AGA GCA CAG TAA TCT TCC ATC-5 ';

When the TCA, the codon of the 107th serine, is substituted with TGC, the codon of the cysteine, the sense primer 5'-TAC AAT ACT TAC CGG TGC AGG AAA TAC ACC AGT-3 'and the antisense primer 3'-ACT GGT GTA TTT CCT GCA CCG GTA AGT ATT GTA-5 ';

Senseprimer 5'-GGA CCT GGG CAG AAA TGC ATA CTT TTT CTT CCA-3 'and antisense primer 3'-TGG AAG AAA AAG TAT GCA TTT CTG CCC when GCT, the codon of alanine, is substituted with TGC, the codon of cysteine AGG TCC-5 ';

Senseprimer 5'-CCT GGG CAG AAA GCT TGC CTT TTT CTT CCA ATG-3 'and antisense primer 3'-CAT TGG AAG AAA AAG GCA AGC TTT when ATA, the codon of 136th isoleucine, is substituted with TGC, the codon of cysteine CTG CCC AGG-5 ';

Senseprimer 5'-GGG CAG AAA GCT ATA TGC TTT CTT CCA ATG TCT-3 'and antisense primer 3'-AGA CAT TGG AAG AAA GCA TAT AGC TTT CTG CCC-5 '; And

Sense primer 5'-TTT CTT CCA ATG TCT TGC AAG AGC TGA TGA-3 'and antisense primer 3'-TCA TCA GCT CTT GCA AGA CAT TGG AAG AAA when GCT, the codon of the 143th alanine, is substituted with TGC, the codon of cysteine -5 '.

Sense primer 5'-GAG AAG AGC GAC CCT TAT ATC AAG CTA CAA CTT-3 'and antisense primer 3'-AAG TTG TAG CTT GAT ATA AGG GTC GCT CTT CTC-5 '.

Example 3: Production and Purification of bFGF Variants

Expression of bFGF Variant Incubated in 500 ml LB medium (LB / ampicillin) and purified in the same manner as in Example 1 to obtain bFGFs each having a size of about 18 KDa. At this time, the amount of the mutant was different depending on the mutant, and about 4 to 12 mg of bFGF was obtained according to the mutant, and the purity was more than 98%.

Each bFGF variant is as follows.

A variant wherein the 68th and 86th cysteines of SEQ ID NO: 1 are substituted with serine and the 33rd isoleucine and the 66th glycine are substituted with cysteine.

A variant wherein the 68th and 86th cysteines of SEQ ID NO: 1 are substituted with serine and the 33rd isoleucine and 69th alanine are substituted with cysteine.

A variant wherein the 68th and 86th cysteines of SEQ ID NO: 1 are substituted with serine and the 33rd isoleucine and 83rd alanine are substituted with cysteine.

A variant wherein the 68th and 86th cysteines of SEQ ID NO: 1 are substituted with serine and the 39th valine and 81st leucine are substituted with cysteine.

A variant wherein the 68 th and 86 th cysteines of SEQ ID NO: 1 are substituted with serine and the 39 th valine and 83 th alanine are substituted with cysteine.

A variant wherein the 68th and 86th cysteines of SEQ ID NO: 1 are substituted with serine and the 49th histidine and 68th cysteine are substituted with cysteine.

A variant wherein the 68th and 86th cysteines of SEQ ID NO: 1 are substituted with serine and the 51st lysine and 67th valine are substituted with cysteine.

A variant wherein the 68th and 86th cysteines of SEQ ID NO: 1 are substituted with serine and the 75th methionine and 107th serine are substituted with cysteine.

A variant wherein the 68th and 86th cysteines of SEQ ID NO: 1 are substituted with serine and the 116th alanine and 135th alanine are substituted with cysteine.

A variant wherein the 68th and 86th cysteines of SEQ ID NO: 1 are substituted with serine and the 116th alanine and 136th isoleucine are substituted with cysteine.

A variant wherein the 68th and 86th cysteines of SEQ ID NO: 1 are substituted with serine and the 74th alanine is substituted with cysteine.

A variant wherein the 68th and 86th cysteines of SEQ ID NO: 1 are substituted with serine and the 25th lysine and the 86th cysteine are substituted with cysteine.

A variant wherein the 68th and 86th cysteines of SEQ ID NO: 1 are substituted with serine and the 137th leucine is substituted with cysteine.

A variant wherein the 68th and 86th cysteines of SEQ ID NO: 1 are substituted with serine and the 51st lysine and 143th alanine are substituted with cysteine.

A variant wherein the 68th and 86th cysteines of SEQ ID NO: 1 are substituted with serine, the 74th alanine is substituted with cysteine, and the 49th histidine is substituted with tyrosine.

Purification of the native and variants can be accomplished through SP-sepharose and heparin affinity columns. Final heparin affinity column purification was followed by SDS-PAGE analysis.

As shown in FIG. 3, the dimer and trimer were observed in the natural form, whereas the variant (HsbFGF) was found to exist in the form of a single band in the monomer size. This data shows that inactive dimers and trimers are completely removed and present in monomeric state.

Experimental Example 1: Structural Analysis Using Circular Dichroism of Natural and Variant bFGF

The structure and thermal stability of the purified bFGF variant of Example 3 were measured by circular dichroism analysis using J-810 spectropolarimeter (JASCO). Natural bFGF was used bFGF purified in Example 1. For structural analysis, each bFGF is dissolved in 20 mM sodium phosphate (pH 7.0) to make a final concentration of 0.1 mg / ml. And then put in 0.1 cm cell, band width 1 nm, response 0.25 sec, data pitch 0.1 nm, scanning speed 20 nm / min, cell length 1 cm, accumulation No. 8, temperature at 20 ℃ Was analyzed.

To analyze the thermal stability, Tm was determined by comparing far-UV at 20 ℃ and 95 ℃ to determine the wavelength of 208 nm and proceeded to 0.1 mg / ml concentration in 0.1 cm cuvette. The conditions were measured from 20 degreeC to 95 degreeC on condition of 1 degree-C / min. The results are shown in Table 1.

변이체 bFGF 명칭Variant bFGF Name	구조변화 (Tm)Structural change (Tm)	변이체 bFGF 명칭Variant bFGF Name	구조변화(Tm)Structural change (Tm)	변이체 bFGF 명칭Variant bFGF Name	구조변화(Tm)Structural change (Tm)
본 발명의 bFGF(서열번호 1)BFGF of the present invention (SEQ ID NO: 1)	- (57.5℃)-(57.5 ℃)	변이체 AC68S,C86SI33C,G66C(서열번호 3)Variant AC68S, C86SI33C, G66C (SEQ ID NO: 3)	감소48℃48 ℃	변이체 BC68S,C86SI33C,A69C(서열번호 4)Variants BC68S, C86SI33C, A69C (SEQ ID NO: 4)	변화 없음No change
변이체 CC68S,C86SI33C,A83C(서열번호 5)Variant CC68S, C86SI33C, A83C (SEQ ID NO: 5)	변화 없음No change	변이체 DC68S,C86SV39C,L81C(서열번호 6)Variant DC68S, C86SV39C, L81C (SEQ ID NO: 6)	변화 없음No change	변이체 EC68S,C86SV39C,A83C(서열번호 7)Variant EC68S, C86SV39C, A83C (SEQ ID NO: 7)	변화 없음No change
변이체 FC68S,C86SH49C(서열번호 8)Variant FC68S, C86SH49C (SEQ ID NO: 8)	변화 없음No change	변이체 GC68S,C86SK51C,V67C(서열번호 9)Variants GC68S, C86SK51C, V67C (SEQ ID NO: 9)	변화 없음No change	변이체 HC68S,C86SM75C,S107C(서열번호 10)Variants HC68S, C86SM75C, S107C (SEQ ID NO: 10)	변화 없음No change
변이체 IC68S,C86SA116C,A135C(서열번호 11)Variant IC68S, C86SA116C, A135C (SEQ ID NO: 11)	변화 없음No change	변이체 JC68S,C86SA116C,I136C(서열번호 12)Variant JC68S, C86SA116C, I136C (SEQ ID NO: 12)	변화 없음No change	변이체 KC68S,C86SA74C(서열번호 13)Variant KC68S, C86SA74C (SEQ ID NO: 13)	변화(62℃)Change (62 ℃)
변이체 LC68S,C86SL25C(서열번호 14)Variant LC68S, C86SL25C (SEQ ID NO: 14)	변화 없음No change	변이체 MC68S,C86SK137C(서열번호 15)Variant MC68S, C86SK137C (SEQ ID NO: 15)	변화 없음No change	변이체 NC68S,C86SK51C,A143C(서열번호 16)Variant NC68S, C86SK51C, A143C (SEQ ID NO: 16)	변화 없음 No change
변이체 OC68S,C86SA74C,H49Y(서열번호 17)Variant OC68S, C86SA74C, H49Y (SEQ ID NO: 17)	(65℃)(65 ℃)

Table 1 shows the results of measuring unfolded fraction by temperature at wavelength of 208 nm during circular dichroism analysis, which is a measure of the degree of structural change and thermal stability of native bFGF and bFGF variants. When the fold-unwinding phenomenon occurs, the structural change is shown in the region around 208 nm, and the accurate Tm value was analyzed by measuring the Tm within the range of 20 ~ 95 ℃.

In Table 1, SEQ ID NO: 1 is a variant in which the methionine is added as a starting sequence after removing two amino acid sequences of the N-terminal sequence from the natural sequence.

As a result of measuring the Tm of the novel variant produced in the present invention, compared to the results of the existing patent (KR 10-2015-0078930 / PCT-KR2015-007734), it shows a Tm pattern similar to the existing HsbFGF, the N-terminal sequence Shows little effect on the structure of bFGF.

As shown in the existing patents, most of the structural changes were identical to those of the natural type bFGF and did not have a special structure in the variants added with disulfide bonds. In the case of Tm, which exhibits thermal stability, it was shown to be almost similar to the experiment of the existing patent, and almost the same as the bFGF variant compared to the natural type bFGF at 58 ° C, among which the variant K (K74) ) Tm value was increased to 62 ℃, Tm showing the thermal stability of variant O (new HsbFGF) substituted for tyrosine 49th histidine in variant K was increased to 65 ℃. Based on these experiments, it was confirmed that the thermal stability was improved and the data were consistent with the contents of the existing patent.

Experimental Example 2 Assay for Cell Proliferation of Natural and Variant bFGF

Cell proliferation assay was performed by selecting bFGF which showed good results through analysis of structure and Tm using solubility and circular dichroism among natural bFGF and variants. Cell proliferation assays were performed with the NIH-3T3 cell line, a skin cell sensitive to bFGF. As an experimental method, NIH-3T3 cells were maintained using DMEM complete medium containing 10% heat-inactivated fetal bovine serum, 100 units / ml penicillin, and 100 mg / ml streptomycin. Seed the NIH-3T3 cells of 2x10 ³ cells / well in a 96 well culture plate. NIH-3T3 cells incubated for 24 hours were treated with serum-free DMEM medium, and then sample solution was treated in DMEM medium containing 0.5% FBS for each concentration, followed by incubation for 72 hours. After incubation, 10 ul of MTT [3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl-2H-tetrazolium bromide] solution was added and reacted for 2 hours, followed by a formazan crystal with 100 ul of DMSO. Was dissolved. Absorbance was measured at 540 nm using a spectrophotometer. Drug susceptibility was compared as a percentage of drug-treated wells for absorbance of untreated drug (control).

Experimental Example 3: Quantitative Analysis of Proteins According to Incubation of Natural and Variant bFGFs

In order to confirm the stability of the natural type bFGF and variants, 37 ℃, 50 ℃, 60 ℃ incubation test was carried out. In the PBS (phosphate buffer) state, which is the most similar to the human state, natural bFGF and its variants were dissolved at 0.5 mg / ml and incubated in a water bath at each temperature. Proteins were quantified using nano drop by sampling at 0, 24, 48 hours, 7 days and 10 days and centrifuging at 13,000 rpm for 15 minutes at 4 ° C. Over time, the concentrations of native bFGF and variants were quantified, and the native bFGF decreased more severely than the variants.

In addition, based on the above results, incubation of the existing HsbFGF, new HsbFGF variant and native bFGF with increased thermal stability was performed. As a result, the variants were much more stable than the native bFGF. Based on these experiments, the natural type, the existing HsbFGF, and the new HsbFGF were incubated at 37 ° C for one month.

Experimental Example 4: SDS-PAGE and HPLC Analysis of Long-Term Incubation of Natural and Variant bFGFs at 37 ° C>

In order to confirm the stability of the natural bFGF and variant K and the novel HsbFGF variant, an incubation test was conducted at 37 ° C. for 100 days. Each natural bFGF and its variants were dissolved in PBS (phosphate buffer) at 0.5 mg / ml and incubated in a 37 ° C water bath. Fractions were taken according to the date and centrifuged at 13,000 rpm for 15 minutes at 4 ° C. to obtain only supernatants. Proteins were analyzed using HPLC and SDS-PAGE.

As shown in Figures 11 and 12, the new HsbFGF was confirmed that the SDS-PAGE and HPLC analysis is preserved only after 100 days.

Experimental Example 5: L929 Killing Analysis

Mouse L929 cells (ECACC, no. 85011425) were maintained using DMEM complete medium containing 10% heat-inactivated fetal bovine serum, 100 units / ml penicillin and 100 mg / ml streptomycin. Seed the NIH-3T3 cells of 5x10 ³ cells / well in a 96 well culture plate. L929 cells were incubated for 24 hours, sample solution was treated for each concentration, and incubated for 24 hours. After incubation, 10 ul of MTT [3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl-2H-tetrazolium bromide] solution was added and reacted for 2 hours, followed by a formazan crystal with 100 ul of DMSO. Was dissolved. Absorbance was measured at 540 nm using a spectrophotometer. Drug susceptibility was compared as a percentage of drug-treated wells for absorbance of untreated drug (control). As can be seen in Figure 13, freshly prepared new HsbFGF was not detected toxicity.

Experimental Example 6: N-terminal Analysis

Edman N-terminal analysis was performed on the purified protein through the above procedure at Korea Institute of Mass Spectrometry. Prepare to be 0.5 mg / ml. Soak PVDF membrane in 100% MeOH for 30 seconds to 1 minute. Then transfer using 0.45 um PVDF membrane. Immerse the blotting finished membrane in tertiary distilled water for 5 minutes. Dyeing is done within 5 minutes using dyeing reagent. After discarding the dyeing reagent, bleaching is performed immediately using a bleaching reagent. When the band of protein is clearly visible, discard the decolorizing reagent and wash it with 3rd distilled water. Air dry the membrane after washing. Since the initial reaction of N- terminal polypeptide chains is formed through phenylisothiocyanate (PITC) coupling reaction, stabilization of phenylthiocarbamyl (PTC) -polypetide, anilinothiazolamine (ATZ) -amino acid and final phenylthiohydantoin (PTH) derivative amino acid The structure is formed and the sequence is checked sequentially by injecting the amino acid derivative generated in each repetition into HPLC. In the case of the PITC peaks shown in FIGS. 8 and 9, the remaining PITC reagent remains during the coupling of the N-terminal amino acid with the PITC reagent, which is shown in the mass spectrometry, which is not related to the N-terminal amino acid. In addition, Dptu in the following peak is a reagent used as a reference in separation using chromatography as a reference and standard reagent.

Claims

A highly stable basic fibroblast growth factor (bFGF) variant, wherein at least two amino acids in the amino acid sequence of SEQ ID NO: 1 are substituted with serine and at least one amino acid is substituted with cysteine.
The variant according to claim 1, wherein the variant induces disulfide bonds in the molecule by substituting the 68th and 86th cysteines of SEQ ID NO: 1 with serine, and then replacing other residues with cysteine.
According to claim 1, wherein the amino acid substituted with the cysteine, in the amino acid sequence of SEQ ID NO: 1 25 lysine, 33 isoleucine, 39 th valine, 49 th histidine, 51 lysine, 74 th alanine, 75 th mesio Ninth, 116th alanine, 66th glycine, 67th valine, 69th alanine, 81th leucine, 83rd alanine, 107th serine, 135th alanine, 136th isoleucine, 137th leucine and 143th alanine High stability bFGF variant, characterized in that at least one member selected from the group.
The bFGF variant of claim 1, wherein the 68th and 86th cysteines of SEQ ID NO: 1 are substituted with serine, the 74th alanine is substituted with cysteine, and the 49th histidine is substituted with tyrosine.
Gene encoding the variant of any one of claims 1 to 4.
The gene according to claim 5, wherein the gene consists of a DNA base sequence of SEQ ID NO.
An expression vector comprising the DNA base sequence of claim 5.
A transformant transformed by the expression vector of claim 7.
Highly stable bFGF variant production method comprising the following steps:

(a) culturing the transformant of claim 8; And

(b) separating the variant from the culture obtained in step (a).
The method of claim 9, wherein step (b) comprises:

(c) cell disrupting the transformant and removing aggregates;

(d) separating and purifying the supernatant from which the aggregates have been removed using ion exchange resin chromatography; And

(e) separating and purifying the highly stable bFGF variant after ion exchange resin using heparin affinity chromatography.
A cosmetic composition for improving skin condition comprising the highly stable bFGF variant of any one of claims 1 to 4 as an active ingredient.
A pharmaceutical composition for preventing or treating skin diseases comprising the highly stable bFGF variant of any one of claims 1 to 4 as an active ingredient.